Human neutrophils, alone, did not kill Trichomonas vaginalis. More than 90% of T. vaginalis (10(5)/ml) survived in the presence of 10% normal human serum (NHS) while 90% of these organisms were killed in the presence of a combination of neutrophils (10(6)/ml) and 10% NHS. Mechanisms responsible for this serum-mediated neutrophil killing of T. vaginalis were demonstrated through a process of lucigenin-amplified neutrophil chemiluminescence. As evidenced by indirect immunofluorescence, NHS showed specific immunoglobulin G (IgG) titre of 1:8 for T. vaginalis. Purified IgG, at 1.6 mg/ml, showed no direct opsonizing or lytic effect on this organism. Formalin-fixed trichomonads opsonized by C2 deficient human serum promote 4 times more neutrophil chemiluminescence than those opsonized by Factor B deficient human serum. With the addition of purified IgG (5 mg/ml) neutrophil chemiluminescence was increased by 4 times and further improved trichomonal killing by neutrophils (from 5 +/- 4% to 78 +/- 16%) via activation of the classical complement pathway, but did not alter that due to activation of the alternative complement pathway. These studies indicate that both an IgG-enhanced classical complement pathway activation and an antibody-independent alternative complement pathway activation provide opsonin (C3) for T. vaginalis to facilitate the neutrophil killing mechanism.
We have investigated the effects of a novel neutrophil-activating factor released by Trichomonas vaginalis (TV-NAF) on neutrophil chemotaxis. TV-NAF was present in the supernatant from 107 T. vaginalis (STV) cultured in 1 ml of serum-free Hanks' balanced salt solution (HBSS) at 37°C for 30 min. With a multichamber chemotactic assay, we found that there were 112 + 15 migrated neutrophils (mean ± standard deviation, n = 7) for STV and 11 ± 4 for HBSS per high-power field (x400). STV was also able to induce neutrophil actin assembly (increased 1.5-fold), enhance expression of complement receptor type 3 (increased 5-fold), and promote intracellular calcium mobilization (increased 2.5-fold). There was no chemotactic activity in the preparation of STV from killed trichomonads. The fact that heating up to 100°C or deproteinization by treatment with proteinase K at 65°C for 1 h did not abolish its chemotactic activity suggests that the TV-NAF involved was not a protein. The chemotactic activity of TV-NAF was associated with the fraction containing small molecules of less than 3,000 Da. Therefore, the possibility that eicosanoid production by trichomonads is responsible for neutrophil activation was investigated. Leukotriene B4 (LTB4; 500 pg/ml) but not thromboxane B2 (<20 pg/mi) or prostaglandin E2 (<8 pg/ml) was found in the STV by radioimmunoassay. Production ofLTB4 by trichomonads was time dependent and increased twofold when arachidonic acid (100 ,uM) was added but was not decreased when eicosanoid inhibitors were present. Evidence for the presence of LTB4 in STV was further provided by the fact that rabbit anti-LTB4 antiserum could abolish the chemotactic activity of STV. These studies suggest that the spontaneous release of TV-NAF, which is most likely LTB4, may activate neutrophils, presumably through a different arachidonate metabolic pathway than that in mammalian cells. * Corresponding author. cellular calcium mobilization. The results from these studies provide more information about the inflammatory response induced by T. vaginalis. MATERIALS AND METHODSOrganism. Seven local isolates, axenically cultivated, were maintained in a modified medium identical to the TYI-S-33 medium of Diamond et al. (7), except that 0.5% Panmede (Paines & Byrne Limited, Greenford, England) was added. The number of organisms per culture was determined with a Coulter counter (model D industrial; Coulter Electronics, Inc., Hialeah, Fla.) with a 70-,um aperture tube.Preparation of STV. Each isolate of T. vaginalis grown at 37°C in 15-ml tubes containing TYI-S-33 medium was harvested during the logarithmic growth phase after 36 h of cultivation. They were centrifugally washed three times in HBSS (GIBCO, Grand Island, N.Y.), and then viable cells were counted in a hemacytometer with trypan blue-saline. HBSS without phenol red was used as the wash buffer and diluent throughout this study. The medium-free flagellates (107) were incubated in 1 ml of HBSS at 37°C for 30 min. Motile flagellates were then removed by gentle centrifugation (500 ...
Complement activity in 125 cases of classical dengue fever was examined through the measurement of haemolytic activity. During the first 3 d of fever, the classical complement pathway activity (CCPA) was not altered in 109 cases. After 4 d of fever, 9 of 16 patients in the viraemic period had CCPA decreased by 45% (995 +/- 119 units/ml) and serum complement component C4 decreased by 40% (10.4 +/- 0.9 mg/dl). The alternative complement pathway activity was not affected in any case tested throughout both viraemic and convalescent stages. Both CCPA and C4 persistently decreased in 3 of these 9 patients at the convalescent stage. A decrease in serum C3 was also observed in these 3 patients only, and circulating immune complexes (CIC) levels were particularly high in these 3 patients. These results indicate that there is little evidence of complement activation on days 1-3 of viraemia but that complement activation may occur subsequently. It is concluded that both CIC and other unknown factors not related to CIC may contribute to complement activation in some cases (9/125) of classical dengue fever.
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